Fig. 5
The m6A demethylase FTO negatively regulates the osteogenesis of BMSCs stimulated with AGEs. A Immunofluorescence assay showed the protein level and location of FTO in negative control (shNC) and FTO-knockdown (shFTO) BMSCs. Scale bars = 25 μm. B, C RT-qPCR and WB analysis of FTO expression in negative control (shNC) and FTO-knockdown (shFTO) BMSCs treated with or without AGEs (n = 3). D M6A dot blot analysis revealed the m6A methylation levels in BMSCs infected with shFTO under the exposure to AGEs or not. Methylene blue stain was utilized as loading control. E Representative images of ALP and ARS staining and quantification of ALP activity and mineralization nodules in BMSCs infected with shFTO under the exposure to AGEs or not after osteogenic induction for 7–21 days (n = 3). Scale bars = 400 μm. F RT-qPCR analysis of the expression of Runx2, Bglap, Col1a and SOST in BMSCs infected with shNC or shFTO under the exposure to AGEs or not after 3 days of osteogenic induction (n = 3). G Western blot analysis of the expression of Runx2, Bglap and SOST in BMSCs infected with shNC or shFTO under the exposure to AGEs or not after osteogenic induction for 5 days (n = 3). H Immunofluorescence assay showed the protein level and location of FTO and SOST in BMSCs infected with shNC or shFTO under the exposure to AGEs or not. Scale bars = 25 μm. Data are expressed as the mean ± SEM. ns, not significant. *P < 0.05. **P < 0.01. ***P < 0.001. ****P < 0.0001